The esophageal epithelium is an exquisitely regulated epithelium with equilibrium between proliferation, differentiation and apoptosis. As a prototype stratified squamous epithelium, it is a vehicle to understand normal homeostasis, regenerative biology and malignant transformation. Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human squamous cell cancers. ESCC tumors often have a dense accumulation of extracellular matrix (ECM) proteins within them, a phenomenon known as desmoplasia. While alterations in critical oncogenes and tumor suppressor genes are common to squamous cell cancers, the extent of desmoplasia may serve as a distinguishing feature. The transformed epithelial compartment and the mesenchymal compartment consisting of stromal fibroblasts, endothelial cells, myocytes and other cell types constitute the microenvironment of the tumor. Signals from tumor cells activate a dynamic process of ECM remodeling that creates an environment conducive for tumor growth. Specialized tumor activated fibroblasts (TAP) have been identified to mediate this remodeling process and promote tumor progression. Using an in vivo-like organotypic 3D cell culture system, we are able to recapitulate classic features of ESCC. This work has led us to hypothesize that activated fibroblasts surrounding the tumor cells help create an environment primed for tumor growth and invasion via the secretion of paracrine growth factors. The interrelated Specific Aims to test this hypothesis and to determine the molecular mechanism(s) by which mesenchymal stromal fibroblasts promote tumor growth and invasion are as follows: (1) To characterize the importance of activated mesenchymal stromal fibroblasts in promoting esophageal cell tumorigenicity. (2): To identify the molecular mechanism(s) by which activated mesenchymal stromal fibroblasts increase the invasive potential of transformed esophageal cells into the surrounding tumor microenvironment. These studies will be done in 3D culture systems and animal models using molecular and cellular biological approaches. In aggregate, our studies will provide new insights into how tumor activated fibroblasts influence squamous cell tumorigenesis, using esophageal cancers as a paradigm, and these findings may unravel innovative combinatorial therapies for treating esophageal cancer through the targeting of both transformed epithelial and mesenchymal stromal cells. Lay language: Esophageal tumors have a dismal prognosis, the worst of any cancer type. It is important to understand how they develop, how different compartments communicate and apply this knowledge to therapy.